CN104755091B

CN104755091B - Selection and use of lactobacilli for preventing osteoporosis in mammals

Info

Publication number: CN104755091B
Application number: CN201380029676.7A
Authority: CN
Inventors: 埃蒙·康诺利; 罗伯特·艾伦·布里顿; 劳拉·雷·麦凯布
Original assignee: Goddess Of Life And Earth Ltd; Michigan State University MSU
Current assignee: Goddess Of Life And Earth Ltd; Michigan State University MSU
Priority date: 2012-06-04
Filing date: 2013-06-04
Publication date: 2020-07-21
Anticipated expiration: 2033-06-04
Also published as: KR20200044134A; EP2854826B9; CA2875841C; US20190142879A1; CN104755091A; AU2018200524A1; CA2875841A1; ES2847424T3; WO2013184064A1; US10537598B2; US9968643B2; DK3456334T3; HK1211847A1; PL2854826T3; EP3456334B1; EP2854826B1; KR102191537B1; DK2854826T3; US20150150917A1; AU2013272330A1

Abstract

The present invention includes a method of selecting a lactic acid bacterium strain effective for preventing osteoporosis in a human body and the strain selected according to the method. The selection method is based on the ability of the strain to reconstitute a variant microbial community to normal and/or to have at least one of four specific SNPs.

Description

Selection and use of lactobacilli for preventing osteoporosis in mammals

Technical Field

The present invention relates generally to medicine, pharmacology and food supplements, and more particularly to the selection and use of lactobacilli for the prevention of osteoporosis in mammals.

Background

Americans over the age of 4000 ten thousand and over 50 years old (of which 1400 thousand are male) suffer from low bone density or osteoporosis and the associated high risk of bone fracture. People with osteoporotic fractures are prone to depression, dependency and increased mortality. Aging is a major cause of osteoporosis, and disease, disuse and certain drugs can also cause osteoporosis at any stage of life.

Bone is a highly organized system that supports the weight of the body, contains bone marrow stromal and hematopoietic stem cells, and serves as a calcium-containing reservoir. The structure of the bone comprises an outer cortical dense shell and an inner cancellous bone mesh. Movement may increase trabecular Bone Mineral Density (BMD), Bone Volume Fraction (BVF), trabecular bone thickness, cortical BMD, and thickness. In contrast, disease, disuse and certain drugs (e.g. glucocorticoids) decrease these parameters and lead to osteoporosis in both men and women. Osteoporosis is defined as a decrease in bone mass (above 2.5 Standard Deviations (SD) below the mean) and altered bone microarchitecture (such as decreased trabecular thickness). The risk of fracture increases with decreasing bone mass. Thus, when diagnosed as osteoporosis, the patient has a 16-fold increase in fracture risk compared to a person with normal bone density. Fractures are associated with depression, dependence and increased mortality (over 25% in 12 months for the elderly), and hip fractures result in over 50000 deaths per year (statistics of the National Osteoporosis Foundation (NOF)). While osteoporosis is less common in men, more than 30% of hip fractures occur in men, and mortality is greater in men than in women. Currently, the united states costs over 200 billion dollars, and the european union costs 300 billion dollars to pay for the immediate cost of osteoporosis. More importantly, it is estimated that by 2020, men and women over the age of 6100 ten thousand 50 in the united states will suffer from low bone density or osteoporosis (NOF statistics), and therefore, the urgent need exists for effective, novel treatments. In fact, one third of women over the age of 50 will experience bone fracture-related osteoporosis in their lifetime. With the increased risk of fractures associated therewith, osteoporosis may negatively affect metabolism and insulin secretion. Despite the availability of available treatments on the market, the number of patients with osteoporosis is on the rise in the united states and around the world. There are several reasons for this, including lack of awareness that people are at risk in early life, an increased aging population, and patient non-compliance due to unnecessary drug side effects. Furthermore, traditional treatments for osteoporosis are not always effective. Currently, there is no alternative or natural therapy available to replace osteoporosis drugs for people with low bone density or osteoporosis. Therefore, doctors are looking for new ways to increase the bone density of patients and companies are striving to improve pharmacological bone treatment drugs.

Some people develop osteoporosis more easily than others, and the risk factor is;

female

Senior citizen

Family history of osteoporosis or broken bones

Short and thin

Some races/ethnicities, e.g. caucasian, asian, or spanish/latin america

Humans, although non-American countries are also at risk

History of bone fracture

Low sexual hormone level

Low estrogen levels include those in women in menopause,

those with menostaxis (amenorrhea)

Male with low testosterone and estrogen levels

Food and drink

Low calcium uptake

Low vitamin D intake

Excessive intake of protein, sodium and caffeine

Lazy life style

Cigarette with suction cup

Excessive drinking

Certain drugs such as steroid drugs, anticonvulsant drugs, and the like,

certain diseases and conditions such as anorexia nervosa, rheumatoid arthritis, gastrointestinal disorders, and the like,

women in menopause are more prone to osteoporosis during menopause due to decreased estrogen levels. Estrogen levels may begin to decline even during menopause (during the first 2 to 8 years of menopause). Over time, massive osteoporosis leads first to a decrease in bone mass (low bone mass) and then to osteoporosis.

The diagnosis of type 1 diabetes (T1D) in children and adults is increasing. Although medical advances extend the life of patients, maintaining normal blood glucose is still difficult even under vigilant treatment. Thus, more patients with T1D (both male and female) suffer from complications including osteoporosis. This means that the patient begins to age/menopause with an already increased risk of fracture. Once a fracture occurs, they are difficult to heal, require long hospitalizations, reduce quality of life and increase mortality. Poor bone health also negatively affects the entire body. Postmenopausal women with T1D diabetes have a higher incidence of osteoporotic fractures than women without diabetes. Children with T1D have lower bone density than children without diabetes. Therefore, maintaining bone health is crucial to the overall quality of life of T1D patients, and is very important for maximizing medical/curative therapy involving bone marrow immune/progenitor cells due to bone marrow cell and bone marrow cell interconversions.

Patients with type II diabetes (T2D) also have a higher risk of osteoporotic fractures than non-diabetic patients.

Two key elements in strengthening bone and preventing osteoporosis are: 1) maximum bone density is achieved and 2) osteoporosis is prevented during adulthood and during aging. Bone remodeling occurs because bone is dynamically changing and continually adapts to environmental cues to form or resorb bone. Targeted bone remodeling is maintained at critical ranges of blood calcium levels by the action of osteoblasts (bone-forming cells) and osteoclasts (bone-resorbing cells), while keeping the bone strong at the sites where support is needed. When the build and resorption activities are balanced, there is no net increase or decrease in bone, however, when build is decreased and/or resorption is increased, osteoporosis ensues.

The cells produce cells of the monocyte/macrophage lineage that develop into precursors of osteoclasts under appropriate conditions, and signal osteoclast maturation by factors such as RANK L (located on the surface of osteoblasts). the mature osteoclasts express enzymes involved in bone matrix degradation (including cathepsin K and TRAP5 b).

Increased osteoblast activity leads to bone formation, which can be regulated at multiple levels, including 1) selection of lineages, 2) maturation and 3) death because Bone Marrow Stromal Cells (BMSCs) produce osteoblasts, adipocytes and other cell types, the selection of one lineage (adipocytes) may be at the expense of another lineage (osteoblasts). the reciprocal relationship between bone fat and mineral density supports this theory, which is recognized by aging, limb offloading, cell culture models, and type I diabetes (T1). the activity of osteoblasts can be further regulated by death/apoptosis.

However, many of these compounds require fasting administration, may cause gastric reflux and nausea, leading to reduced patient compliance, concern also about the length of time these compounds remain on the bone, and their long-term effects on bone remodeling and bone Strength.alternative Estrogen Receptor Modulators (SERMS) are another therapeutic treatment, but they may present problems for cancer.

Disclosure of Invention

The main object of the present invention is to provide a method how to find lactic acid bacterial strains capable of preventing osteoporosis, especially climacteric women, diabetic patients, osteopenic persons, including for example young people with a large energy intake and a low exercise frequency.

It is an object of the present invention to use a product comprising said strain in climacteric women for the prevention of osteoporosis.

It is an object of the present invention to use a product comprising said strain in women who have undergone hysterectomy, in order to prevent osteoporosis.

Another object is to use products containing said strains in men, including but not limited to diabetics, young men with metabolic disorders and osteopenic men, for the prevention of osteoporosis.

Another object is to combine said product with a therapeutic drug for osteoporosis or bone formation in order to reduce the dosage of said drug, thus enabling to minimize the side effects.

Another object is to improve bone repair after fracture.

Accordingly, a first aspect of the present invention provides a method of selecting a lactobacillus strain for the prevention or treatment of osteoporosis, the method comprising selecting a lactobacillus strain having at least 95% identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and having the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), said nucleotides being located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment according to the first aspect, the method comprises selecting a lactobacillus strain having at least 96% (such as 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and said lactobacillus strain having the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

A second aspect of the invention provides a method of selecting a Lactobacillus strain for the prevention or treatment of osteoporosis, such as a Lactobacillus reuteri strain, the method comprising selecting a Lactobacillus reuteri strain having the same nucleotides, relative to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1), located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the method according to the first or second aspect, the lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the four nucleotides.

A third aspect of the present invention provides a method of selecting a Lactobacillus strain for preventing or treating osteoporosis, the method comprising selecting a Lactobacillus strain having at least 95% identity with respect to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1), with the proviso that the Lactobacillus strain does not have at least one mutation with respect to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to A in base pair 453538, G to A in base pair 529228, and C to T in base pair 599338.

In an embodiment of the third aspect, the method comprises selecting a lactobacillus strain having at least 96% (such as 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), with the proviso that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the method according to the third aspect, the lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

A fourth aspect of the present invention provides a lactic acid bacterial strain for use in the prevention or treatment of osteoporosis, the lactic acid bacterial strain being selected according to the method of the first, second or third aspect.

In an embodiment of the fourth aspect, the lactobacillus strain selected for the prevention or treatment of osteoporosis is lactobacillus reuteri ATCC PTA 6475. The strain is publicly available from the American type culture Collection (university of Manassas, Va., street No. 10801) and is deposited at the type culture Collection under the Budapest treaty at 21.12.2004.

According to a fifth aspect, the present invention provides a lactobacillus strain for preventing or treating osteoporosis, having at least 95% identity with the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and having the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), said nucleotides being located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the fifth aspect, the lactobacillus strain has at least 96% (such as 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) which are located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the fifth aspect, the lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to a sixth aspect, the present invention provides a lactobacillus strain having at least 95% identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), with the proviso that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the sixth aspect, the lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the sixth aspect, the lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

According to a preferred embodiment of the fifth or sixth aspect, said lactobacillus strain is lactobacillus reuteri ATCCPTA 6475.

A seventh aspect of the invention provides a composition comprising a lactobacillus strain selected according to the method of the first, second or third aspect of the invention.

According to an eighth aspect, there is provided a composition comprising a lactobacillus strain having at least 95% identity relative to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and having the same nucleotides relative to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), said nucleotides being located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the eighth aspect, the lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the eighth aspect, the lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to a ninth aspect, the present invention provides a composition comprising a lactobacillus strain having at least 95% identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), with the proviso that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the ninth aspect, the lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the ninth aspect, the lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, such as any of the four mutations.

In a preferred embodiment of the eighth or ninth aspect, the lactobacillus strain is lactobacillus reuteri ATCCPTA 6475.

In an embodiment of the eighth or ninth aspect, the composition is for use in the prevention or treatment of osteoporosis.

In another embodiment of the eighth or ninth aspect, the composition is for use in preventing osteoporosis in menopausal women, women with hysterectomy, diabetic patients, osteopenic individuals, osteoporotic individuals, and metabolic disorders.

In a further embodiment of the eighth or ninth aspect, the composition is for improving bone repair after fracture.

In an embodiment of the eighth or ninth aspect, the above composition is used in combination with vitamin D for the prevention or treatment of osteoporosis.

In another embodiment of the eighth or ninth aspect, the above composition is used in combination with a hormone (for hormone replacement therapy) for the prevention or treatment of osteoporosis.

In an embodiment of the eighth or ninth aspect, the above composition is a pharmaceutical composition (preferably comprising at least one pharmaceutically acceptable excipient), or a food product or food supplement (preferably comprising at least one food grade excipient, which is well known to the person skilled in the art).

According to a tenth aspect, the present invention provides the use of a lactobacillus strain having at least 95% identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and having the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), said nucleotides being located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338 for the preparation of a pharmaceutical composition for the prevention or treatment of osteoporosis.

In an embodiment of the tenth aspect, the Lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1) and the same nucleotides with respect to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1) are located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the tenth aspect, the lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to an eleventh aspect, the present invention provides the use of a lactobacillus strain having at least 95% identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), with the proviso that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), said mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338, for the preparation of a pharmaceutical composition for the prevention or treatment of osteoporosis.

In an embodiment of the eleventh aspect, the lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the eleventh aspect, the lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

In a currently preferred embodiment of the tenth or eleventh aspect, the lactobacillus strain is lactobacillus reuteri ATCC PTA 6475.

A twelfth aspect of the invention provides a method for treating or preventing osteoporosis, the method comprising administering to an individual a Lactobacillus strain having at least 95% identity relative to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1) and having identical nucleotides relative to the genome of Lactobacillus reuteri JCM1112(SEQ ID NO: 1) located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the method according to the twelfth aspect, the lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) and the same nucleotides with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1) are located in at least one of the following four positions: c in base pair 271391, G in base pair 453538, G in base pair 529228, and C in base pair 599338.

In an embodiment of the twelfth aspect, the lactobacillus strain has at least two of the four nucleotides, such as at least three of the four nucleotides, all four of the nucleotides.

According to a thirteenth aspect, the invention provides a method for treating or preventing osteoporosis, the method comprising administering to an individual a lactobacillus strain having at least 95% identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), with the proviso that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the thirteenth aspect, the lactobacillus strain has at least 96% (e.g. 97%, 98%, 99%) identity with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), provided that the lactobacillus strain does not have at least one mutation with respect to the genome of lactobacillus reuteri JCM1112(SEQ ID NO: 1), the mutation being selected from the group consisting of the four mutations C to T in base pair 271391, G to a in base pair 453538, G to a in base pair 529228, and C to T in base pair 599338.

In an embodiment of the thirteenth aspect, the lactobacillus strain does not have at least two of the four mutations, such as at least three of the four mutations, any of the four mutations.

In a currently preferred embodiment of the twelfth or thirteenth aspect, the lactobacillus strain is lactobacillus reuteri ATCC PTA 6475.

Drawings

FIG. 1 shows the microflora which accumulate in the jejunum and ileum;

FIG. 2 shows the inhibition of osteoporosis by Lactobacillus reuteri ATCC PTA 6475;

FIG. 3 shows the effect on osteoporosis in different Lactobacillus reuteri strains.

Detailed description and preferred embodiments of the invention

Chronic inflammatory diseases are often associated with systemic osteoporosis. In the abstract of grant No. 1R21AT005472-01a1, granted by the National Institute of Health (NIH), mikebab indicates that therapies that can improve overall intestinal health potentially have beneficial bone health. Mikebab and brillton found that lactobacillus reuteri therapy was able to reduce the levels of TNF in the ileum, increase bone volume in healthy male mice but not female mice, and suggested that lactobacillus reuteri increased bone density in a gender-dependent manner by inhibiting intestinal inflammation and upregulating bone formation. They believe that lactobacillus reuteri has a new approach to increase bone mass by using probiotics that reduce intestinal inflammation. However, unlike certain specific strains selected in the present invention, which are sex-dependent upregulated in bone formation, rather than prevention of osteoporosis, which is associated with the anti-inflammatory properties of Lactobacillus reuteri, certain specific strains selected in the present invention for prevention of osteoporosis in men and women are different.

The probiotics can increase the thickness of chicken cortical bone and reduce osteoporosis of aged mice. Narva et al describe the effect of lactobacillus helveticus fermented milk on osteoporosis in ovariectomized mice of "bioactive peptides, valyl-prolyl-proline (VPP) and lactobacillus helveticus fermented milk containing VPP", which may be due to the effect of the peptide valyl-prolyl-proline. Narva et al further describe the positive acute effect of Lactobacillus helveticus fermented milk on calcium metabolism in the "effect of Lactobacillus helveticus fermented milk on acute changes in calcium metabolism in postmenopausal women".

Yeo et al, in "angiotensin I-converting enzyme inhibitory activity and bioconversion of isoflavones by probiotics in prebiotic-supplemented soymilk" indicate that probiotics mixed in prebiotic-supplemented soymilk can potentially be used as a dietary treatment, such as osteoporosis.

Kim et al showed that lactobacillus casei 393FMP has a preventive effect on osteoporosis in ovariectomized mice in "effect of lactobacillus casei 393 in fermented milk products on bone metabolism in ovariectomized mice".

However, the above mentioned prior art does not give any hint how to select specific probiotic bacterial strains for effective prevention of osteoporosis, either alone or in combination.

The invention herein includes a method of selecting a strain of lactobacillus that is effective in preventing osteoporosis in a human and the strains selected according to the method. Products such as foods, nutritional supplements and formulations, pharmaceuticals or medical devices containing all cells or components from these strains can be prepared, and typically include an ingestible support known to have added lactobacillus strains, or components derived therefrom, as is known in the art.

Based on the prior art, one can naturally think that the capacity of a strain to prevent osteoporosis is linked to its general effect on intestinal health or its anti-inflammatory properties, however, the inventors have surprisingly found that these properties are unpredictable for the effect of preventing osteoporosis. Lactobacillus reuteri ATCC PTA6475 and lactobacillus reuteri ATCC PTA4659 are two nearly identical strains, both of which are anti-inflammatory and improve gut health. Therefore, it is naturally assumed that these strains also exert the same effect on osteoporosis. However, the inventors found that these strains do not have the same effect on preventing osteoporosis, and based on this observation, they invented a novel method for selecting a lactic acid bacterium strain such as lactobacillus reuteri, which will effectively treat and/or prevent osteoporosis.

The lactobacilli specifically selected according to the methods provided herein are administered to humans to prevent osteoporosis.

Lactobacillus reuteri ATCC PTA6475 and lactobacillus reuteri ATCC PTA4659 differ at four SNPs that are important for the ability of the bacteria to prevent osteoporosis. These SNPs are shown by Walter et al (Walter et al, in the vertebrate gastrointestinal tract host-microorganism symbiosis and Lactobacillus reuteri paradigm; Proc. Natl. Acad. Sci. USA, 108 vol. 4645 page 4652), the entire contents of which are incorporated herein by reference. For SNP analysis, the sequencing results were mapped to the reference genome (Lactobacillus reuteri JCM1112, GenBank accession AP007281, SEQ ID NO: 1). The 7 SNPs were found in lactobacillus reuteri ATCCPTA4659, 3 of these 7 SNPs were also found in lactobacillus reuteri ATCCPTA6475 (SNP 4 located at bp 567368, SNP6 located at bp 968088, and SNP 8 located at bp 1358460, with respect to the reference genome, lactobacillus reuteri JCM1112, GenBank accession No. AP007281, SEQ ID NO: 1), and the remaining 4 unique SNPs (for the purposes herein, hereinafter SNP1, SNP2, SNP3, and SNP 5, respectively) constituted the genomic differences between lactobacillus reuteri ATCC PTA6475 and lactobacillus reuteri ATCC PTA 4659. The four SNPs are located:

-bp 271 391(SNP 1)，

-bp 453 538(SNP2)，

bp 529228 (SNP3), and

-bp 599 338(SNP5)，

(for the reference genome, Lactobacillus reuteri JCM1112, GenBank accession number AP007281, SEQ ID NO: 1).

SNP1 is located at the gene coding for a conserved putative protein (Lactobacillus reuteri JCM 1112: http:// www.ncbi.nlm.nih.gov/protein/183224225), SNP2 is located at the gene coding for chloride channel protein (Lactobacillus reuteri JCM 1112: http:// www.ncbi.nlm.nih.gov/protein/183224386), SNP3 is located at the gene coding for the gamma subunit of ATP synthase (Lactobacillus reuteri JCM 1112: http:// www.ncbi.nlm.nih.gov/protein/183224455), and SNP 5 is located at the gene coding for the DNA mismatch repair protein HexB (Lactobacillus reuteri JCM 1112: http:// www.ncbi.nlm.nih.gov/protein/183224511). The SNP related to the invention is a SNP matched with Lactobacillus reuteri ATCCPTA6475, and the sequence of the SNP is the same as that of the Lactobacillus reuteri JCM1112 at the positions of SNP1, SNP2, SNP3 and SNP 5. Listed below are the nucleotides that differ between lactobacillus reuteri ATCC PTA6475 and 4659:

SNP1) was used for the gene encoding the putative protein, in which nucleotide 267 was changed from C (e.g., ATCCPTA6475 and JCM1112) to T in ATCCPTA 4659.

SNP 2) for the gene encoding chloride channel protein, where nucleotide 373 was changed from G (e.g., ATCC PTA6475 and JCM1112) to a in ATCC PTA 4659.

SNP3) for a gene encoding the ATP synthase gamma subunit, wherein nucleotide 296 was changed from G (e.g., ATCC PTA6475 and JCM1112) to a in ATCC PTA 4659.

SNP 5) was used for the gene encoding the HexB protein, in which nucleotide 1966 was changed from C (e.g. ATCC PTA6475 and JCM1112) to T.

In the selection method of the invention, the strain sought is one in which at least one of these SNPs has the same nucleotide as the above-mentioned SNP of lactobacillus reuteri ATCC 6475 PTA.

The microbiota plays an important role in osteoporosis; many patients with osteoporosis have a disturbed intestinal microbiota. Lactobacilli, which are capable of reconstituting a normal microbial community in the gastrointestinal tract, are surprisingly more effective in preventing osteoporosis.

The present invention discloses a unique selection method for selecting strains effective for the prevention of osteoporosis. The ability to reconstitute the total intestinal microbial composition is also surprisingly very important for the function of preventing osteoporosis. The inventors found that strains capable of reconstituting an altered microbial community as normal and/or as strains having at least one of four specific SNPs are effective for preventing osteoporosis.

The ability to prevent osteoporosis is unique to certain strains, and not all lactobacillus strains have this unique ability. In selecting effective strains, it is not sufficient to use anti-inflammatory capacity as a selection criterion, since the inventors have shown specifically that this effect is not dependent on anti-inflammatory properties. Both lactobacillus reuteri ATCC PTA6475 and lactobacillus reuteri ATCC PTA4659 are anti-inflammatory strains, but lactobacillus reuteri ATCC PTA6475 is more effective when used to prevent osteoporosis, whereas lactobacillus reuteri ATCC PTA4659 is not selected according to the present invention. In general, the particular lactobacillus strains selected according to the present invention may be used to prevent osteoporosis, and the following examples are not intended to limit the scope of the present invention, but to exemplify preferred embodiments.

Vitamin D is very important for bone health, and people with low vitamin D levels have lower bone density and bone quality. Because vitamin D is required for calcium absorption, people who do not ingest sufficient vitamin D may develop osteoporosis. The inventors have found that altered microbiota can lead to vitamin D deficiency and osteoporosis and administration of lactobacilli selected according to the invention will reestablish microbiota, thereby increasing the intestinal absorption of vitamin D and restoring vitamin D levels. Combining vitamin D with the selected strain is also a choice in order to obtain a more efficient method/product for preventing osteoporosis.

Patients with T1D suffer from complications such as osteoporosis. Patients with T1D will therefore have an altered microbiota. Administration of lactobacilli selected according to the present invention will reestablish microbiota and prevent osteoporosis.

High bone density in young and adult stages may help prevent disease of osteoporosis in later years. This is because a high bone density will allow a higher degree of osteoporosis before the bone density in the osteoporotic region is reached. Therefore, the object of the present invention is to prevent osteoporosis by administering the lactobacillus strains selected according to the present invention to young and adults, which can help individuals to obtain maximum bone density to prevent osteoporosis from occurring later in life. The particular lactobacilli selected according to the present invention may prevent osteoporosis in healthy recipients as well as in those suffering from osteoporosis.

Administration of the selected lactobacillus may be combined with hormone replacement therapy. Such a combination enables a reduction in the amount of hormone used, thereby reducing side effects, such as a reduced risk of developing cancer.

The lactic acid bacterium selected for the prevention of osteoporosis is preferably administered to menopausal women and men who are susceptible to osteoporosis, and the administration of the selected lactic acid bacterium can prevent osteoporosis, thereby preventing low bone density and osteoporosis.

The inventors have appreciated that estrogen consumption alters gut microbiota and treatment with lactobacilli selected according to the invention will reestablish microbiota in patients with reduced estrogen levels, thereby preventing osteoporosis and reducing estrogen levels

The lactic acid bacterial strains selected according to the invention may also be used for improving fracture repair.

To reduce the side effects of drugs, such as diphosphate and hormone replacement therapy for the treatment of osteoporosis, drugs may be combined with the administration of selected lactobacilli to reduce the dosage and minimize side effects.

Example 1

Study of the ability of lactobacillus reuteri ATCC PTA6475 to reconstitute altered microbial communities in ovariectomized mice.

Controls (no ovariectomized), ovariectomized, and ovariectomized fed with lactobacillus reuteri had significant changes in the intestinal microflora.

Experimental groups and tissue Collection

To measure the effect of ovariectomized mice (ovx) and ovariectomized mice treated with lactobacillus reuteri 6475, we compared three experimental animal groups. Control mice, which were not ovariectomized mice, received 3 solvent control feedings per week. Ovariectomized mice received 3 solvent control feedings per week. Mice spayed with ovariectomized and lactobacillus reuteri 6475 received 3 μ l of overnight lactobacillus reuteri 6475 weekly for 4 weeks. At the end of the experiment, mice were euthanized and tissue samples from the stomach, duodenum, jejunum, ileum, proximal colon and distal colon were isolated and stored for microbial ecological analysis.

DNA extraction

Mouse intestinal tissue was placed in a MoBio ultra clean fecal DNA magnetic bead tube (cat. # 12811-.

Polymerase chain reaction amplification

Bacterial 16S sequences were amplified for 454 sequencing from mouse intestinal tissue using a V3-V5 barcode primer set and an amplification protocol developed by the human microbiology project broudder research institute. Barcode forward primer was synthesized by IDT DNA technology and reverse primer was synthesized by Sigma. The barcode forward primer was diluted to a working concentration of 4 μ M in 96-well plates; reverse primers were added to each well at a final concentration of 4. mu.M. A25. mu.l volume of the reaction was prepared in triplicate, the reaction comprising 400. mu.g mouse gut DNA, 2. mu.l of 4 μm primer and 0.15. mu.l Accuprime HiFi Taq polymerase in 1 XAccuprime buffer II (Invitrogen, cat. # 12346086). The reaction was amplified in an Eppendorf (Eppendorf Pro) aluminum plate thermal cycler with 2 min 95 ℃ denaturation followed by 30 cycles of 95 ℃ x 20 sec, 50 ℃ x 30 sec, 72 ℃ x 5 min.

Amplification of product purification

The 16S amplification product was purified using Ampure Agencourt XP magnetic beads (Beckman Coulter, cat # A63880). First, three reactions of each sample were mixed in a 1.7 ml microcentrifuge tube and Ampure XP magnetic beads were added at a 0.7-fold volume ratio. After vortexing, the mixed samples were incubated at room temperature for 10 minutes and then placed on a magnetic watch for magnetic bead separation (Invitrogen, cat. # 123-21D). The beads were washed 2 times with 200. mu.l 70% ethanol according to the manufacturer's instructions. The magnetic beads were dried at 37 ℃ for 5 minutes, and the DNA was eluted with 20. mu.l of 10. mu.M Tris buffer (Tris)/0.1. mu.M ethylenediaminetetraacetic acid (EDTA). The eluate was separated from the magnetic beads on a magnetic stand and transferred to a new 1.7 ml microfuge tube for quantification using a Quant-It dsDNA high sensitivity assay kit (Invitrogen, cat # Q33120). Equal amounts of each sample were then pooled into 454 sequencing tubes.

454 sequencing and sequence analysis

454 sequencing was performed using GS Primary (Roche) using titanium chemistry. In addition to using the standard filter of the GS primary to determine the determined sequences that have been delivered, we used a modified amplicon processing algorithm to reduce erroneously discarded sequences. Alignment of 16S rRNA sequences at MSU and organization at E.coli 16S nucleotide positions 617 to 900 was performed by ribosomal database project personnel relative to E.coli 16S sequences. Subsequent processing and analysis (including diversity index) were performed using MOTHUR v.1.21 (http:// www.mothur.org/wiki /). ANOSIM (similarity analysis) and principal coordinates analysis were performed using the software package PAST. The degree of dissimilarity between two or more microbial communities was measured using the Bray-Curtis method using the figures and tables. In these analyses, we selected the operational taxon (OUT) with a cutoff value of 0.03, which is considered to be the colony observed at the seed level. From these data, we conclude that treatment of ovariectomized mice fed lactobacillus reuteri ATCC PTA6475 produced a significant shift in the microflora in the ileum and jejunum associated with improving bone health. (ovariectomized and lactobacilli in Table 1).

TABLE 1

Tissue of	Control	R value (p value)
			Jejunum	Wild type-ovariectomy with lactobacillus feeding	0.3367(0.0183)*
	Wild type-ovariectomy	0.0443(0.3633)
				Wild type-ovariectomized lactobacillus feeding	0.6078(0.0250)*
	Ovariectomy-ovariectomy with lactobacillus feeding	0.3297(0.0712)
			Ileum	Wild type-ovariectomy with lactobacillus feeding	0.2068(0.0084)*
	Wild type-ovariectomy	0.1710(0.1180)
				Wild type-ovariectomized lactobacillus feeding	0.2540(0.0290)*
	Ovariectomy-ovariectomy with lactobacillus feeding	0.2209(0.0206)*

Table 1: ANOSIM analysis was performed at the seed level using the Bray-Curtis dissimilarity matrix,

indicates statistical significance

Three-way comparisons of wild type, ovariectomy and ovariectomy with treatment with lactobacilli showed significant changes in the microflora (table 1). these differences were largely due to the large changes in the microflora after treatment with lactobacillus reuteri.major coordinate analysis of the microflora from the wild type control group (triangle △), the oophorectomized group (circle ○) and the oophorectomized group treated with lactobacillus reuteri (square □) was used to see how the microflora accumulated in the jejunum and ileum figure 1 shows that the ovariented mice treated with lactobacillus reuteri from one cluster of the microflora are significantly different from the microflora in the jejunum and ileum of wild type and ovariectomized mice.

Example 2

The experiment was carried out as in example 1, but with the replacement of Lactobacillus reuteri ATCC PTA6475 by Lactobacillus reuteri ATCC PTA 4659.

Lactobacillus reuteri ATCC PTA4659 therapy failed to reconstitute ovariectomized mice relative to control.

Example 3

Identification of specific SNPs

Illumina sequence of Lactobacillus reuteri genome

The Lactobacillus reuteri used in this study were ATCC PTA4659 and 6475 grown in MRS medium and Genomic DNA was prepared using the Genomic-Tip system. The DNA was lysed by 20 min sonication (130W) to obtain fragments of an average of 500bp, which were then further purified and concentrated using QIAquick PCR purification spin columns (Qiagen). The treatment to remove 3 'extension and fill in 5' extension results in blunting of the ends of the genomic fragment. Adenine fragments were added to the 3' terminus by terminal transferase, and the fragments thus generated were ligated to a Solexa adapter. The products were separated by agarose gel electrophoresis and fragments between 150bp and 200bp were excised from the gel. The DNA fragments were extracted from the agarose gel slides using the QIAquick gel extraction kit (Qiagen). Adapter-modified DNA fragments were enriched by 18-cycle PCR using Solexa universal adapter primers. The pool of DNA fragments was quantified and then diluted to 10-nM working stock for colony clustering. Adapter-ligated fragments (2nM) were denatured in 0.1M sodium hydroxide solution for 5 min, then further diluted to a final concentration of 9pM in 1 ml of pre-chilled hybridization buffer and concentrated and introduced onto Solexa flow cells using Cluster Station. Following isothermal amplification, the flora clusters were made single stranded by 0.1M sodium hydroxide denaturation and the flow cells were metered through by Solexa Cluster Station. Sequencing primers complementary to the Solexa adapters were added to single strands of each cluster. Once the excess primers are hybridized and eluted, the flow of cells is ready for sequencing. Solexa genome analyzer II was programmed to provide 36 serial flows of fluorescent labels, 3' -OH to block nucleotides and polymerase onto the surface of the flow cells, resulting in a fixed read length of 36 bp. After each base incorporation step, the reaction on the surface of the flow cell is eluted and then imaged by a microscope objective. 300 tiled images ("tiles") were collected per flow cell channel in this experiment, each containing an average of 30000 colonies.

SNP analysis

Results of the bidirectional sequencing were mapped to control genome lactobacillus reuteri JCM 1112T (GenBank accession No. AP007281), respectively. Mapping software Maq version 0.6.6 (http:// maq. sourceforce. net/maq-man. shtml) was used for mapping (default parameters). SNPs were identified and verified with MAQ software and classified as coding SNPs and intergeneric SNPs. The encoded SPNs are determined to be synonymous and non-synonymous. After Sanger sequencing, SNPs were finally verified by PCR amplification of the peripheral region.

Example 4

Selection method of strain

The selection of strains effective in preventing osteoporosis is based on the ability to reconstitute altered microbial communities. Based on the results of examples 1 and 2, lactobacillus reuteri ATCC PTA6475 was selected based on the fact that the strain has the ability to reconstitute altered microbial communities. Lactobacillus reuteri ATCC PTA4659 was not selected based on the results of example 2.

Example 5

Selection method of strain

The selection of strains that are effective in preventing osteoporosis is based on the presence of specific SNPs. From the results of example 3, lactobacillus reuteri ATCC PTA6475 was selected because it has all four desired SNPs. Lactobacillus reuteri ATCC PTA4659 was not selected because it lacks these SNPs.

Example 6

Selection method of strain

Selection of strains effective in preventing osteoporosis is based on examples 3, 4 and 5, selecting strains with at least one of the four desired SNPs and having the ability to reconstitute altered microbial communities. Lactobacillus reuteri ATCC PTA6475 was selected based on these criteria.

Example 7

Lactobacillus reuteri ATCC PTA6475 inhibits ovariectomized induced osteoporosis

In this study, ovariectomized (ovx) BA L B/c mice were used as a mouse model for osteoporosis, mice (12 weeks old) were ovariectomized, divided into two groups, the first group was treated with Lactobacillus reuteri ATCC PTA6475 3 times per week for 4 weeks, the non-ovariectomized BA L B/c was used as a control group, distal femoral bone volume fraction (BV/TV) and bone TRAP5RNA (vs. HPRT) were determined, the bone volume fraction of mice treated with Lactobacillus reuteri ATCC PTA6475 was the same as the control group, in addition, TRAP5 (a marker of osteoclast function) was also seen to return to baseline (control group) on the basis of Lactobacillus reuteri ATCC PTA6475 treatment.

Figure 2 shows that lactobacillus reuteri ATCC PTA6475 inhibited osteoporosis by nearly 100%, and that the expression of TRAP5 returned to baseline.

Example 8

The selected lactobacillus reuteri ATCC PTA6475 is superior to the unselected lactobacillus reuteri ATCC PTA4659 in inhibiting osteoporosis.

In this experiment we fed the animal strains lactobacillus reuteri ATCC PTA6475 and lactobacillus reuteri ATCC PTA4659 in a fill-feed manner, while also providing uninterrupted provision of the strains in drinking water for 28 days. Distal femoral bone volume fraction (BV/TV) was measured by μ CT. Lactobacillus reuteri ATCC PTA6475 inhibits osteoporosis and is indistinguishable from control mice (fig. 3). Lactobacillus reuteri ATCC PTA4659 failed to inhibit osteoporosis to a sufficient level to reach statistical significance (p <.01). Lactobacillus reuteri ATCC PTA4659 is not as effective as the selected Lactobacillus reuteri ATCC PTA 6475.

Claims

1. Lactobacillus reuteri: (L reuteri) Use of ATCC PTA6475 in the manufacture of a composition for the prevention or treatment of osteoporosis.

2. Use according to claim 1 for the prevention of osteoporosis in menopausal women, women with hysterectomy, diabetic patients, osteopenic individuals, osteoporotic individuals and metabolic disorders.

3. Use according to claim 1 for improving bone repair after fracture.

4. The use according to claim 1, wherein the composition is used in combination with vitamin D.

5. The use according to claim 1, wherein the composition is used in combination with a hormone.

6. The use of any one of claims 1-5, wherein the composition is a pharmaceutical composition.